The objectives of this PPG are to understand what controls the choice between tolerance and autoimmunity. Using established experimental models, the component projects will explore the molecular pathways and genetic modifiers of central and peripheral T-cell tolerance to self-antigens. Project 1. Mechanisms of peripheral CD4 T-cell tolerance (PI: A. Abbas). The aims of this project are to define: 1. signaling abnormalities and the roles of known immune regulators and cytokines (IFN-y, IL-2) in T cell anergy and deletion induced by a transgene-encoded systemic secreted protein, and 2. the mechanisms that control the relative development of effector and regulatory T-cells specific for the systemic self-antigen. Project 2. CTLA-4 function in tolerance and autoimmunity (PI: J. Bluestone). This project will explore the hypothesis that the ligand-binding and ligand-independent forms of the inhibitory receptor CTLA-4 play distinct roles at different stages of T-cell responses. The specific aims are to study: 1. the molecular mechanisms of action of the two forms of CTLA-4, 2. the roles of the two forms of CTLA-4 in cell-intrinsic tolerance, and 3. their roles in preventing lymphoproliferation and maintaining homeostasis. Project 3. Regulation of CD45 signaling in tolerance and autoimmunity (PI: A. Weiss). This project will focus on CD45 as a paradigm for the role of tyrosine phosphatase signaling in maintaining self-tolerance. The specific aims are to study: 1. the influence of an inactivating CD45 wedge mutation on T- and B-cell tolerance, 2. the roles of myeloid cell abnormalities caused by the mutation, 3. genetic modifiers of the autoimmune phenotype, and 4. molecular basis of the regulatory function of CD45, specifically the wedge. Project 4. Control of autoimmunity by central tolerance (PI: M. Anderson). Building on the seminal discovery of the role of the AIRE protein in thymic negative selection, this project will explore: 1. genetic modifiers of autoimmunity caused by AIRE deficiency, 2. interactions between central tolerance and peripheral regulatory mechanisms, and 3. AIRE-mediated self-antigen expression as a therapeutic strategy for autoimmunity. Cores. Core A (animals) will create new genetically modified mouse strains, maintain strains shared by all projects, and assist with breeding and genotyping for genetic studies. Core B (flow cytometry) will provide state-of-the-art multi-parameter analyses and high-speed cell sorting for all projects. Thus, this PPG brings together four highly complementary, interactive and productive investigators to address fundamental questions about the biology and mechanisms of T-cell tolerance and autoimmunity. The proposed studies will lay the foundation for developing new immunological therapies for immune mediated inflammatory diseases. PROJECT 1: Mechanisms of peripheral CD4 T-cell tolerance (Abbas, A.) PROJECT 1 DESCRIPTION (provided by applicant): The overall objective of this project is to define the cellular and molecular mechanisms of CD4 T-cell tolerance to a systemic self-antigen. The studies will test the hypotheses that cell-intrinsic tolerance in T-cells results from a combination of proximal signaling blocks and imbalanced cytokine production, and when the cell-intrinsic mechanisms of anergy and deletion fail, prolonged self-antigen recognition without other stimuli may lead to progressive development of regulatory T-cells. The studies will rely on a well-established transgenic model of systemic T-cell tolerance which has some unique strengths, notably that it is amenable to biochemical and molecular analyses of cells that have encountered self-antigen, and it is the only system in which effector and regulatory T-cells are generated sequentially from a monoclonal T-cell population in response to self-antigen recognition in peripheral tissues. The following specific aims will be addressed: 1. Mechanisms of cell-intrinsic tolerance (anergy and deletion) induced by a systemic self-antigen. These studies will define signaling blocks in T-cells rendered anergic by recognition of systemic self-antigen in vivo, using a novel multiplex phosphoprotein array and other techniques, and examine the roles of known T-cell regulators (CTLA-4, Fas, Bim) in systemic tolerance. In addition, the studies will explore the novel idea that imbalanced production of IFN-y without IL-2 contributes to T-cell tolerance, and define the mechanisms underlying this unexpected role of IFN-y as a tolerance-inducing cytokine. 2. Induction and functions of peripherally generated regulatory T-cells (Treg). Using a model of sequential development of effector T-cells and Treg in response to recognition of systemic antigen, these studies will define the lineage relationships between these two cell populations and the roles of cytokines in controlling the balance between effector and regulatory cells. A model of parent to F1 graft-vs-host reaction will be used to examine the development of effector and regulatory T-cells in situations of polyclonal T-cell reactivity. Thus, this project uses a defined experimental system and a variety of precise analytical methods to study fundamental mechanisms of peripheral T-cell tolerance, its induction and maintenance, and its regulation by external signals. There are numerous close interactions between this project and project 2 (studies of CTLA-4 and Treg), project 3 (signaling pathways), and project 4 (interactions of central and peripheral tolerance mechanisms). The results are not only of biological significance, but will also provide valuable leads for strategies to induce tolerance as a therapeutic modality.